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This adenovirus is part of abm’s Adenoviral Expression System and can be used directly to transiently over-express your gene of interest in a wide range of host cells. This adenovirus can be used to amplify more adenovirus by transducing HEK293 cells. For enhanced transduction efficiency, the use of ViralPlus (G698) at 1:100 is recommended at the time of transduction.

High titer adenoviruses are shipped with dry ice and low titer adenoviruses are shipped with ice packs. For long term storage, it is recommended to store the viruses at -80°C in small aliquots to avoid repeated freeze-thaw cycles.

Disclaimer

1. Disclaimer for transcript variants: The provided accession number refers to the transcript (mRNA) sequence for this product. The molecular sequence of this clone aligns with the gene accession number as a point of reference only. However, individual transcript sequences of the same gene can differ through naturally occurring variations (e.g. polymorphisms), each with its own valid existence. This clone is substantially in agreement with the reference, but a complete review of all prevailing variants is recommended prior to use. All sales are final.
2. The provided accession number refers to the transcript (mRNA) sequence for this product. Please verify that this is the desired transcript sequence by cross-referencing. This is important because a single gene can have multiple different transcripts owing to naturally occurring variations. All sales are final.
3. Disclaimer for intended use: All of abm's vectors and viral particles are for research use ONLY and NOT for therapeutic/diagnostic applications. abm is not liable for any repercussions arising from the use of its vector(s) in therapeutic/diagnostic application(s).
4. Disclaimer for extra nucleotides: Cloning may lead to the insertion of extra nucleotides at the 5' or 3' end of the gene of interest which, in most cases, is innocuous to the stability/functionality and the expression of that gene.

Guarantee

abm guarantees that the correct ORF construct is provided and the mRNA expression is displayed upon successful transduction. If this is not the case, we will provide a one-time replacement. Customers must provide adequate data to show >80% transfection efficiency with a positive control, plus additional qPCR data to evaluate the level of gene expression. The replacement will not be covered by the same guarantee.

Please note that due to the large number of variables applicable, any further expression analysis (e.g. protein expression) is not covered by the guarantee, as such analysis is dependent on the end user's experimental conditions.

The pAdeno system is a 2nd generation vector derived from human adenovirus type 5.

Which adenoviral vector is used for the construction of the pre-made Adeno-plasmids?

Human adenovirus 5

How is the titer determined to be 10^6 cfu/ml?

We produce the virus via an established procedure (standardize packaging mix and expression vector amount) that will give a minimal titer of 1X10^6 pfu/ml.

Does it require the purchase of additional products to produce adenovirus? If so what are the necessary related products?

Customers need 293 cells to amplify adenoviral constructs.

What is the required biosafety level for using recombinant adenovirus?

The recombinant adenoviruses are replication deficient due to deletions in the E1 and E3 regions. According to references issued by the NIH Office of Biosafety, recombinant human adenovirus has been classified in biosafety level II for agents considered of ordinary potential harm, and you need BL-2 level facility to work with it. It should be noted that cell culture facilities in most institutes are certified as BL-2 level. Wild type, replication competent adenoviruses could cause cold symptoms but generally do not cause serious illness. For more information on biosafety levels please visit http://bmbl.od.nih.gov

Our primary experimental model is the mouse. Will this system work for infecting primary mouse cells?

Most adenoviral vectors are human based adenovirus which will give 100% transduction efficiency in human cells except blood cells. Most mouse cells are also transducible with 100% efficiencey with adenoviral vectors, but some of them are not transducible due to a lack of adenovirus receptors on their cell surface. A good way to solve this problem is to use a marker adenovirus like GFP to determine whether the cell posses the receptor or not before beginning a big project.

How do I check the efficiency of adenovirus infection in my target cells?

The Adenovirus has a very broad host range; it can infect human and other mammalian cell lines or primary cells, including dividing and non-dividing cells. There are in fact very few cell lines that cannot be infected. It has been shown that cells of Hematopoietic origin are more resistant to adenovirus infection, and so may need high quantities of viruses to achieve sufficient infection levels as those cells have only limited CAR receptors expressed on cell membrane.

For your convenience, we offer some marker adenoviruses, such as Ad-CMV-beta-Gal or Ad-GFP to allow you to evaluate infection efficiency in your target cells.

What's the optimal concentration of viruses for infection?

The appropriate MOI (multiplicity of infection) of recombinant viruses used for infecting cells is very important for the outcome of your experiments. If MOI is low, it will not give 100% of infection. If MOI is too high, it will cause cytotoxicity or other undesired effects. You should use the minimal virus concentration that will give 100% gene delivery. This optimal concentration differs dramatically among different cell types. To determine optimal concentration of virus, you could perform pilot experiments by using marker adenoviruses, such as Ad-beta-Gal or AdEGFP. For most cell types, a viral concentration of of 2 x 10e5- 1 x 10e6 per ml of media gives 100% of infection without visible side effects. For your reference, we recommend the following amount virus-containing media for infection (given that the adenovirus concentration is 1 x 10e6 particles per ml):

10-cm plate: 5 ml per plate6-well plate: 1 ml per well12-well plate: 0.5 ml per well24-well plate: 0.2 ml per well This volume roughly covers the surface area of each well or plate.

For in vitro use (cell culture studies), is viral purification required?

No. If viruses will be used in in vitro cell cultures, double CsCl purification is not required. For in vivo studies (i.e. animal studies), purification is essential in order to remove defective particles, cell debris, and small amounts of media components, since these contaminants induce significant immune responses. In addition, CsCl purification will concentrate the virus to a level suitable for in vivo injections.

What are the recommended storage conditions of recombinant Adenoviruses?

For long-term storage, the virus should be kept at -80C, especially after CsCl or chromatography purification. At -80C in 50 mM Tris (pH 7.0) plus 5% glycerol, the viruses could be stable for several years. Alternatively, viral stock could be stored in 10mM Tris (pH 8.0) plus 4% sucrose for in vivo injection as it is more difficult to do in vivo injection with glycerol formulation.

What are RCAs?

One concern when working with adenoviral vectors is the rare occurrence of replication competent adenoviruses (RCAs) in a population of replication-deficient viruses. RCAs can emerge as a result of the rare double crossover through overlapping sequences present in the recombinant adenovirus and the genome of 293 cells. This event results in the replacement of the transgene by E1 region. Once this happens, the adenovirus could replicate in target cells (non-permissive cells). To detect RCA, non-permissive cells, such as A549 cells, are incubated with the viral stocks and monitored for cytopathic effects (CPE). According to NIH guideline, <1 plaque in about 10^4 viruses is considered safe to use in clinical trials. To avoid the occurrence of RCA, viruses should be produced and amplified in low passage 293 cells.

What is the concentration or titer of the premade adenovirus?

The seed stock is 10^6 pfu/ml; however, the customer can amplify as much as needed in 293 cells.

Viral particles (VPs) represent the total number of viral particles (infectious and infection-deficient combined). Due to variations in virus preparations, the ratio of infectious /non-infectious varies significantly and therefore, VP does not reflect the concentration of virus in a preparation. PFU ( plaque forming unit) represents the number of infectious or live viruses. It reflects the concentration of infectious viruses in a preparation. IFU (infectious unit) is biologically equivalent to PFU. For most virus preps, the VP/PFU ratio is 20:1 to 50:1.

OD260 assay measures the concentration of viral DNA. It does not distinguish intact, infectious viruses from damaged, non-infectious ones. It is a physical assay measuring the number of total viruses, live and dead. Based on OD260, the concentration of viral particles (VP) could be obtained. To measure the OD260, the virus stock has to be purified first.

On the other hand, plaque formation assay measures the concentration of infectious viruses, and therefore it is a biological assay. Basically, a monolayer of 293 cells is infected with a series of virus dilutions from 10^1 to 10^12. Viruses will propagate in infected cells, and eventually cause complete cytotoxicity effects (CPE) in that cell, and get released. The released viruses will infect neighboring cells, and the whole process will be repeated, eventually leading to the formation of holes or plaques on the cell monolayer. In order to prevent the diffusion of viruses among plaques, a layer of agarose is laid on top of cells after initial infection. The biological principle or End-point Dilution assay is similar to the plaque formation assay, although the procedure and measurement is different, and the formula for calculating the virus titer is a bit more complicated.

Although both the Plaque Formation Assay and End-point Dilution assay gives the titer of infectious or working viruses, they are scored by human eyes and subjected to human and procedure variations. For the same virus stock, it is quite common that different people will get significantly different titer readings.

How does TCID50 work?

Please see the link before for the assay. TCID50 is the same as end dilution assay. http://www.abmgood.com/TechSupport/adeno-vec.php

If the customer provide us the virus, we can provide its titer with the Cat#C008 service.

Does the order come with a control or empty virus?

The control or empty vector virus (Adeno CMV-null) is sold separately. You can find this product under the Cat. No. 000047A.

What is the cell density that the customer should use for adenovirus infection?

70%

Is it possible to use the adenovirus directly without amplification in 293 cells? Can we use other cells than 293?

Adenoviruses are supplied as a seed stock of 1x10^6 pfu/ml, and should be amplified before use. If not enough virus is used, it will not give 100% of infection. The Adenovirus has a very broad host range; it can infect human and other mammalian cell lines or primary cells. If you find that your cell line may be more resistant to Adenovirus infection, you may need to use high quantities of the virus to achieve sufficient infection levels.

What cells do I use to amplify the adenovirus?

It must be 293 cells for amplification. 293T cells will not work as well.

Are the His and HA tags placed at the N or C terminus?

All adenovirus His and HA tags are at the C-terminus (unless otherwise specified).

Do you have more information about the insert sequence?

The insert contains the full CDS region of the gene according to the accession no. listed above.

I have received more than 250ul of seed stock and have concerns it has been diluted or is the wrong product entirely.

We occasionally have an excess from production of our adenoviruses and in these cases we choose to distribute it as an additional volume to the stated 250ul for our customer's benefit. Please be assured you have received the correct item, the viral titer will still be 10^6 pfu/ml

I just received 10^6 pfu/ml Adenovirus stock from your company, and amplified according to the protocol provided. What is the estimated final PFU after 3 cycles of freezing-thawing?

The neat supernatant from amplifying the virus will still be 10^6pfu/ml. The virus will need to be concentrated if a higher titer is required.

If you wish to achieve a higher titer, we recommend using one of our purification kits:http://www.abmgood.com/Adenovirus-Purification.html

How do you determine the viral titer of this product?

The viral titer will be calculated by end-point dilution assay. Any additional assays required (Plaque Assay, RCA Assay or TCID50 Assay) can be provided on request, at an additional cost.

What is an easy to transduce cell line for adenoviruses?

293 can be used to check expression, however, transduction will lead to plaque formation therefore a better cell line is MCF7, which are easily transduced by adenovirus and is not effected by adenovirus CPE.

If my cells don't divide, would the adenovirus still be considered to confer transient effects?

Yes, even if your cells do not divide, the adenovirus would still be able to infect your cells.

For the amplification protocol, what volume of media is recommended for a 60mm dish or 100mm dish?

We recommend ~3-4ml per 60mm dish and ~10ml per 100mm dish.

Why is "freeze and thaw" cycle needed for making adenoviruses?

Upon harvesting, up to 70% of all the adenoviral vectors are still in the cells. They can be released by repeated freezing and thawing cycles.

In protocol of pre-made adenovirus amplication,
there is a strange sentence,"by infectiing cells with 10ul for 60mm dish, or 200ul for 100mm dish".
I think one of two volume is a mistake. Can you tell me the right volume for 60mm dish and 100mm dish?

100mm is in fact about 5 X 60mm in area. 30ul to 200ul all should be fine and should not make much difference in the amplification of adenovirus.